Expression And Functional Analysis Of Correlative Enzyme Genes Of Lignin Phenylpropanoid Biosynthetic Pathway In Betula Platphylla

Lignin is an aromatic polymers and macromolecular organic material second to cellulose inside plant, which is mainly present in the secondary thickened plant cell wall. It provides the mechanic support for plant, protects the plant from pathogen invasion. Therefore, the lignin synthesis of terrestrial plant is the one of important characteristics in adapting to the land life. Several decades of research have indicated the mainly biosynthetic paths of the monolignols and demonstrated that lignin content can be engineered. Lignin engineering can improve the processing efficiency of plant biomass for pulping, forage digestibility and biofuels. Plants can cope with large shifts in phydroxyphenyl/ guaiacyl/ syringyl (H/G/S) lignin compositional ratios. There are many enzymes in lignin biosynthesis path, of these, Caffeoyl-Coenzyme A 3-O-Methyltransferase (CCoAOMT) is one key enzyme in lignin biosynthesis pathway. It is believed to be involved in the methylation of caffeoyl CoA to produce feruloyl CoA, the precursor of G lignin units.4-coumarate:CoA ligase (4CL), as the other key enzyme in linking the pathways of phenylpropanoic acid and lignin peculiar synthesis, mainly is be involved in cinnamic acid to produce cinnamic acid CoA, which is control point of lignin synthesis and other phenylpropanes compound metabolisms.The study isolated the full length cDNA of birch actin gene firstly, which is the basis of real-time PCR. The degenerate primers were designed according to the conserved sequence of gene encoding region in different plant actin, RT-PCR was carried out, and the full length cDNA of birch actin gene was amplified by RACE. The full length of birch actin gene is 1785 bp, which encodes 377 amino acids, with a 5'-non coding region of 157 bp,3'-non coding region of 495 bp, and coding region of 1134 bp. The similarity of actin gene sequence of birch with other plant registered in GenBank is over 80%, those of their amino acids sequences is up to 96%, the highly conservation showed that the actin gene was closely correlative to cell life, and the construction analysis and homology modeling were conducted. The cDNA (BplPALl) encoding phenylalanine ammonia-lyase (PAL) were cloned from B. platyphylla by reverse transcription polymerase chain reaction (RT-PCR) and 5'and 3'rapid amplification of cDNA ends (RACE), which contains an open reading frame (ORF) (2322 bp) encoding 773 amino acids. The amino acids sequences contain two functional domains of PAL-HAL and PAL, and the sequence of enzyme active site (GTITASGDLVPLSYIA). Semi-quantitative RT-PCR analysis indicated that the expression of BplPALl genes was different in tissues, of these was higher in xylem than in young leaves and inflorescences. The results suggest that BplPALl genes are differently regulated and expressed in tissues.Secondly, on the basis of the isolations for full length cDNA of CCoAOMT and 4CL of birch(BplCCoAOMT1 and Bpl4CLl), the expression analysis of BplCCoAOMTl and Bpl4CL1 genes of birch was carried out in the level of mRNA by Q-RT-PCR and Northern blot. The result of Northern blot showed that BplCCoAOMT1 gene also had a higher transcription level expression in July and August. Otherwise, the relative expression of BplCCoAOMT1 and Bpl4CL1 genes in various phases of organs and secondary xylem of birch was carried out by Q-RT-PCR. The result showed that the BplCCoAOMT1 gene had a higher expression in secondary xylem from the end of June to the early of July, and reached the peak at the end of July. Just as BplCCoAOMTl gene, the transcription level of Bpl4CL1 gene had a significantly difference at the various development phases. Moreover, the BplCCoAOMTl and Bpl4CL1 genes also had a high expression into the secondary xylem, next to leave, and the lowest in leafstalk.Thirdly, antisense BplCCoAOMT1 cDNA was separately transformed into tobacco (Longjiang911 and SR-1) and birch mediated by Agrobacterium tumefaciens and the constructed vector of p121-BplCCoAOMT1. Tissue culture systems and the program of gene transformation of two varieties of tobaccos (Longjiang 911 and SR-1) were compared. The gene transformation processes of the two varieties of tobaccos were basically the same, which also adopt the disc-leaf transformation by mediated A. tumefaciens. At last, we totally acquired 252 seedlings resisting kanamycin. By the PCR detection of specific primers, there were 115 positive plants of 252. The detection result of histochemistry showed that the S-lignin contents of transgenic positive plants of Longjiang911 decreased. The contents of total lignin of positive plants were lower 39.0% than that of the control in average. The results of analysis of variance showed there is a significant difference between the transgenic plants and the control. In addition, the regeneration system and transgenic program of B. platyphylla were optimized, and we initially inferred that the lignin component of transgenic birch plant made a change. Therefore we can conclude that antisense BplCCoAOMT1 cDNA transformation can affect the lignin biosynthesis of transgenic plant in a sense, and BplCCoAOMT1 gene was engaged in the synthesis of lignin and S-monocase.